Thyroid hormones T3 and T4 regulate human luteinized granulosa cells, counteracting apoptosis and promoting cell survival.

PURPOSE: Fine and balanced regulation of cell proliferation and apoptosis are key to achieve ovarian follicle development from the primordial to the preovulatory stage and therefore assure female reproductive function. While gonadotropins are the major and most recognized regulators of follicle cell growth and function, other factors, both systemic and local, play equally important roles. This work is aimed at evaluating the effects of thyroid hormones (THs) on human granulosa luteinized (hGL) viability. METHODS: Human GL cells derived from assisted reproduction treatments were exposed to T3 or T4. Cell viability was evaluated by MTT assay. Apoptosis was evaluated by the TUNEL assay and active caspase-3 staining. StAR, CYP19A1,Caspase-3, P53 and BAX mRNA were evaluated by real-time PCR. LC3-I/-II, AKT and pAKT were evaluated by western blot. RESULTS: T3 and T4 promoted cell viability in a dose-dependent modality and modulate StAR and CYP19A1 expression. T3 and to a lesser extent T4 mitigated cell death induced by serum starvation by inhibition of caspase-3 activity and expression of P53 and BAX; and attenuate cell death experimentally induced by C2-ceramide. Cell death derived from starvation appeared to be involved in autophagic processes, as the levels of autophagic markers (LC3-II/LC3-I ratio) decreased when starved cells were exposed to T3 and T4. This effect was associated with an increase in pAkt levels. CONCLUSION: From the present study, THs emerge as potent anti-apoptotic agents in hGL cells. This effect is achieved by inhibiting the apoptosis signalling pathway of BAX and caspase-3, while maintaining active the PI3K/AKT pathway.

Chapter 8 Human Oocytes Slow-Rate Freezing: Methodology.

Cryopreservation of human oocytes is an important technique for the treatment of human infertility, as it deals successfully with legal, ethical, and moral issues related to embryo cryopreservation (Coticchio et al., Human Fertil (Camb) 4:152-157, 2001; Tucker et al., Eur J Obstet Gynecol Reprod Biol 113: 524-527, 2004) and maintains reproductive potential in women with diseases or conditions that may compromise reproductive capacity. Here, we describe an oocyte cryopreservation technique involving slow freezing-rapid thawing with differential sucrose concentration (0.2 M in the freezing stage-0.3 M in thawing stage) (Bianchi et al., Reprod Biomed Online. 14:64-71, 2007), describing the technique in detail, from the preparation of the solutions through to the performance of the technique and, finally, to a number of helpful hints for optimum results.